Processing of lesions into DNA repair and checkpoint pathways

将病变处理为 DNA 修复和检查点通路

基本信息

批准号：
10595083
负责人：
MATTHEW J O'CONNELL
金额：
$ 34.79万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10595083
关键词：
Aging Bypass Cancer Biology Catalysis Cell Cycle Cell Cycle Checkpoint Cell Cycle Progression Cell Proliferation Centromere Chromatin Complement Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA lesion DNA replication fork Data Degenerative Disorder Development Ensure Environment Enzymes Event Excision Excision Repair Family Family member Fission Yeast Frequencies Funding Genes Genetic Recombination Genetic Screening Genetic Transcription Genome Genomics Genotoxic Stress Goals Grant Heterochromatin Homologous Gene Human Induced Mutation Invaded Learning Lesion Malignant Neoplasms Mediating Minor Planets Modeling Nonhomologous DNA End Joining Okazaki fragments Organism Paper Pathologic Pathway interactions Phosphotransferases Process Property Proteins RAD52 gene Reaction Regulation Resected Role S phase Saccharomyces cerevisiae Saccharomycetales Signal Transduction Single-Stranded DNA Sister Chromatid Site Source Specificity Surgical incisions Syndrome System Testing Time Type I DNA Topoisomerases Work Yeasts biological adaptation to stress cancer therapy endonuclease experimental study genetic analysis genome integrity homologous recombination in vivo loss of function neoplastic cell novel nuclease nuclease I preservation prevent public health relevance recruit repaired replication stress response restraint scaffold tool

项目摘要

DNA damage comes in many forms that originate from intrinsic and extrinsic sources. These lesions can induce the mutations and genome rearrangements that lead to cancer, aging and degenerative diseases. Particularly pathological lesions are the DNA double stranded DNA breaks (DSBs), as well as collapsed replication forks caused by barriers to replication, that can themselves promote DSB formation. To activate cell cycle checkpoints, mechanisms that allow the time to repair these lesions, ssDNA is generated at these sites in a 5’à3’ direction. The resulting ssDNA that remains has an exposed 3’-OH group, and acts as a landing pad for assembly of checkpoint signaling complexes as well as recombination enzymes that promote invasion into the sister chromatid. Using the fission yeast Schizosaccharomyces pombe as a gene and pathway discovery tool, we identified a family of XPG-related nucleases (XRNs) as the long sought after enzymes that are necessary and sufficient for end resection at DSBs. This consists of the long known Rad2/Fen1 and Exo1 enzymes that also function in Okazaki Fragment maturation and various Excision Repair pathways. The newly identified third member of this family is known as the Asteroid nucleases. These include Ast1 in S. pombe and ASTE1 in humans, but there is no Asteroid homolog in the budding yeast Saccharomyces cerevisiae. Thus, Ast1 enzymes remain poorly characterized compared to Fen1 and Exo1. Studies leading to, and during the initial funding period of this grant, have shown that these XRN nucleases are hierarchically recruited to DSBs, which is dynamic depending on the complement of nucleases. There is further specificity afforded by the direction of transcription at a damaged locus. A considerable body of data also shows that the XRNs are critical at collapsed replication forks, cooperating with several other enzymes that modulate fork stability and processing. Additional experiments in this proposal build on these observations and utilize an armory of new tools to study the processing of these lesions across the genome. We present a thorough analysis of Ast1 to bring the understanding of this conserved enzyme to level commensurate with its long-studied cousins, then determine specificity determinants among them. As these initiating events in DNA damage responses feed into many downstream response pathways, this work has significant impact on the study of the many mechanisms that ensure the integrity of the genome.

DNA损伤有多种形式，源自内在和外部源。这些病变可以诱导导致癌症，衰老和退化性疾病的突变和基因组重排。特别病理病变是DNA双链DNA断裂（DSB），也是折叠的复制叉由复制的障碍造成，这本身可以促进DSB的形成。要激活细胞周期检查点，允许时间修复这些病变的机制，在5'à3方向上生成ssDNA。由此产生的ssDNA剩下的3'-OH组，充当装配垫的降落垫检查点信号传导复合物以及促进入侵姐妹的重组酶染色单体。我们使用裂变酵母菌酸果实Charomyces Pombe作为基因和途径发现工具，我们确定了 XPG相关核武器（XRN）的家族作为必要且充分的酶之后的长气味用于DSB的最终切除术。这是由已知的RAD2/FEN1和EXO1酶组成的，该酶也起作用 Okazaki碎片成熟和各种切除修复途径。新确定的第三名成员家族被称为小行星核。其中包括S. pombe和Aste1中的AST1，但有在酿酒酵母的发芽酵母菌中没有小行星同源物。那，AST1酶保持较差与FEN1和EXO1相比，特征的特征。研究导致该赠款的最初资助期间，已经表明了这些XRN 核酸酶在层次上募集到DSB，这是动态的，具体取决于核的完成。在损坏的基因座的转录方向提供了进一步的特异性。一个相当大的机构数据还表明，XRN在折叠的复制叉中至关重要，与其他几种酶合作该调节叉稳定性和处理。本提案中的其他实验基于这些观察结果并利用一系列新工具来研究整个基因组中这些病变的处理。我们提出一个对AST1的彻底分析，以使对该酶的理解与之相称长期研究的表亲，然后确定特异性决定了它们之间的特殊性。这些启动事件在DNA中损害反应源于许多下游响应途径，这项工作对研究确保基因组完整性的许多机制。